Applications of human pre-B-cell leukemia homeobox 1 (PBX1) gene and related drugs thereof

A gene and drug technology, applied in the use of human PBX1 gene and its related drug fields, can solve the problem that the role of PBX1 gene is not elucidated.

Active Publication Date: 2012-07-04
SHANGHAI GENBASE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of PBX1 gene in the occurrence and development of lung cancer, liver cancer and breast cancer has not yet been elucidated

Method used

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  • Applications of human pre-B-cell leukemia homeobox 1 (PBX1) gene and related drugs thereof
  • Applications of human pre-B-cell leukemia homeobox 1 (PBX1) gene and related drugs thereof
  • Applications of human pre-B-cell leukemia homeobox 1 (PBX1) gene and related drugs thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Preparation of RNAi lentivirus against human PBX1 gene

[0068] 1. Screening for effective siRNA targets against the human PBX1 gene

[0069] The PBX1 (NM_002585) gene information was retrieved from Genbank; an effective siRNA target for the PBX1 gene was designed using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the PBX1 gene, a sequence of 21 bases is obtained starting from every other base, and Table 1 lists 29 effective siRNA target sequences for the PBX1 gene.

[0070] Table 1 is targeted at the siRNA target sequence of human PBX1 gene

[0071]

[0072]

[0073] Aiming at the siRNA target (taking SEQ ID NO: 27 as an example), synthesize a double-stranded DNA Oligo sequence (Table 2) containing AgeI and EcoRI restriction sites at both ends; act on pGCSIL- GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify ...

Embodiment 2

[0091] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of PBX1 gene

[0092] Human lung cancer H1299 cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells, human gastric cancer SGC7901 cells and human pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (the MOI of SGC7901 and H1299 is 10, the MOI of Panc-1, SMMC-7721 and MCF-7 is 20) value, add the virus that the suitable amount embodiment 1 prepares, change culture medium after cultivating 24h, wait for After the infection time reached 5 days, the cells were harvested. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcr...

Embodiment 3

[0100] Example 3: Detection of proliferation ability of tumor cells infected with PBX1-shRNA lentivirus

[0101] Human lung cancer H1299 cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells, human gastric cancer SGC7901 cells and human pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, MOI of H1299 and SGC7901 is 10, MOI of Panc-1, SMMC-7721 and MCF-7 is 20), add appropriate amount of PBX1-shRNA virus, change medium after culturing for 24 hours, and wait for infection After the staining time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2000 / well. ...

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Abstract

The invention discloses applications of a human pre-B-cell leukemia homeobox 1 (PBX1) gene and related drugs thereof. The invention discloses the applications of the PBX1 gene in cancer therapy, cancer diagnosis and drug preparation. The invention also further constructs a human PBX1 gene small interfering ribonucleic acid (siRNA), a human PBX1 gene interfering nucleic acid constructor and a human PBX1 gene interfering slow virus and discloses the applications thereof. The siRNA or the nucleic acid constructor containing the siRNA and the slow virus provided by the invention are capable of specifically restraining the expression of the human PBX1 expression, especially, the slow virus is capable of efficiently infecting target cells to efficiently restrain the expression of the PBX1 gene in the target cell, and further, the growth of cancer cells is restrained, and the apoptosis of cancer cells is promoted. The human PBX1 gene has very important meaning in the cancer therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the use of human PBX1 gene and related medicines. Background technique [0002] RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing phenomenon mediated by double-stranded RNA. RNAi technology has high post-transcriptional silencing efficiency and specificity, and is expected to become a tool for gene therapy of tumor diseases (Izquierdo M. Short interfering RNAs as a tool for cancer gene therapy. Cancer Gene Ther. 2005; 12 (3): 217- 27.). Plasmid or viral vector can manipulate the expression of a 45-50nt hairpin RNA (short hairpin RNA, shRNA) in mammalian cells, shRNA will be automatically processed into small interfering RNA (small interfering RNA, siRNA) in the cell, This in turn triggers gene silencing or expression suppression. Lentiviral vectors have the advantages of large capacity for carrying gene fragments, high transfection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/113C12N15/63C12N15/867A61K48/00A61P35/00
Inventor 韩海雄孙琴顾雪峰沈浩金杨晟瞿红花曹跃琼
Owner SHANGHAI GENBASE BIOTECH CO LTD
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